The R43 Phase of this project aims to develop and demonstrate a capillary isotachophoresis (CITP)-based multidimensional protein separation platform, capable of providing selective analyte enrichment and extremely high resolving power for handling complex protein mixtures prior to mass spectrometry detection. Such selective enhancement toward low abundance proteins can drastically reduce the range of relative protein abundances in complex proteomes, and greatly improve proteome coverage using the proposed CITP/nano-reversed-phase liquid chromatography (nano-RPLC) multidimensional separation platform. The proposed proteome technology will be highly automated and offer robust and high throughput fractionation of intact proteins for top-down proteomics while avoiding analyte dilution and loss in comparison with the current and manually operated proteome processes. By coupling with a nanoscale trypsin membrane reactor, the ultrafast proteolytic digestion of proteins resolved and eluted from the CITP-based multidimensional separations enables direct correlation of the peptides and their sequences with the corresponding proteins and measured protein masses in a single analysis, and greatly facilitates the characterization of post-translational modifications in complex proteomes. Research efforts in the R43 Phase will address early implementation, optimization, and demonstration of the proposed proteome technology using yeast cell lysates as a model proteome system. [unreadable] [unreadable]